Immobilization of biologically active material with glutaraldehyde and polyazaetidine

ABSTRACT

Immobilized biologically active material in particle form is prepared by cross-linking with glutaraldehyde and polyazetidine. An aqueous dispersion or solution of biologically active material is partially cross-linked with glutaraldehyde, a wet pasty mass is recovered by dewatering and the mass is sub-divided into discrete particles. A polyazetidine prepolymer is added before, at the beginning or subsequent to partially cross-linking but prior to subdividing the pasty mass into particles, and the prepolymer is allowed to cross-link.

This application is a continuation in part of copending application Ser.No. 874,141 filed June 13, 1986, now abandoned.

This invention relates to a method for immobilizing biological materialsby cross-linking with polyazetidine and, in a preferred mode, to amethod for converting cell bound enzymes into cell mass enzymeparticles.

BACKGROUND OF THE INVENTION

Immobilized enzyme products, especially immobilized enzyme productsintended for use in a column has been a rapidly growing field as of thedate hereof. Research efforts have been directed towards producingimmobilized enzyme products of ever lower price, better physicalstrength, higher unit activity and of particle size and shapes givingrise to a minimum pressure drop during column operation as well as ahigh particle strength against abrasion. As of the date hereof, workersin the art have made available a substantial number of reasonablysatisfactory methods to immobilize enzymes.

This invention is directed, in a preferred mode thereof, to theconversion of cell bound microbial enzymes into particle formimmobilized enzymes made from the cell mass of the microorganism. Thediscussion of enzyme immobilization hereinafter provided is largelywithin a context of this type of immobilized enzyme product.

On the whole, as the art has advanced, product and method deficiencies,such as non-optimum particle size distribution, lack of control overparticle shape and the cost factor of relatively low product yield, longconsidered to be unimportant defects in the immobilization processbecome major defects, which must be obviated. For example,glutaraldehyde has been employed in commercial practice forcross-linking cell bound enzymes according to the teachings of AmotzU.S. Pat. No. 3,980,521. Yet, glutaraldehyde is not an idealcross-linking agent, for cell bound enzymes at least, reacting only with--NH₂ and --SH groups. The cells of many microorganism species reactpoorly with glutaraldehyde.

A polyazetidine prepolymer may be employed advantageously forcross-linking purposes, such being suggested by Wood et al. U.S. Pat.No. 4,436,813 and by "A Novel Method of Immobilization and Its Use inAspartic Acid Production," Wood et al., Bio/Technology, December 1984,pp. 1081-1084. This Patent and Paper ar incorporated by referenceherein. The polyazetidine prepolymer cross-linking system is more widelyapplicable to immobilization of cell bound enzymes than isglutaraldehyde because cross-linking reactions take place between thepolyazetidine prepolymer and --COOH and --OH groups as well as --NH₂ and--SH groups.

The instances to which practice of this invention is directed inparticular are those when the desired enzyme form constitutes particlesmade from the microorganism cells, and cellular substances, andcross-linking reagent(s), and optionally, auxiliary cross-linkingagents, e.g., proteins and/or agglomerating agents, e.g.,polyelectrolytes, and/or finely-divided filler materials. The particlesare essentially homogeneous. Such enzyme product form are variouslytermed herein as cell mass particles and/or cell mass particulate form.It is noted parenthetically, that the process of above-referenced Woodet al. Patent and Paper is directed principally to immobilizing theenzymatically active microorganism cells and cellular substances oncarrier particles, and that the inventors hereof strongly prefer thecell mass particle form over a carrier base particle form of immobilizedenzyme product the latter not being essentially homogeneous particles.

Efforts by the inventors hereof to employ polyazetidine prepolymercross-linking to generate cell mass particulate form enzyme productsevidenced existence of material deficiencies to the process taught bythe prior art. The reaction mixture constitutes an aqueous dispersion ofthe polyazetidine prepolymer in solution and individual microorganismcells along with any cellular substances present, or other biologicalmaterial necessitating conduct of the curing reaction en mass. Bycrushing the reaction product and sieving, a desired particle sizefraction may be recovered, but overall the yield of the wanted particlesize fraction is usually low, and also, the shape of the individualparticles is not controlled. Thus, cross-linking a non-particulate cellmass composition gives rise to immobilized enzyme product whereinparticle shape and size is not controlled.

Although the foregoing discussion of the background of the invention andthe description of the invention which now follows is couched in termsof cell bound enzymes and a cell mass particulate product, such is doneto facilitate understanding of the invention and to describe preferredpractice of the invention in fulsome fashion. It is emphasized thatpractice of the invention is applicable to biological materials moregenerally, including notably, homogenized cell sludge, enzymes insolution (e.g., extra cellular enzymes), co-enzymes and anti-bodies.

OBJECT OF THE INVENTION

The object of the invention is the provision of a method adapted toproduce a particulate form of biological material in high yield, ofparticles with high physical strength, whereby also the shape and sizeof the particles can be controlled.

Enzymes, soluble and cell bound alike, are preferred biologicalmaterials and cell mass immobilized enzyme products are particularlypreferred products of the invention.

Enzymes of particular interest to the inventors hereof are glucoseisomerase, penicillin acylase and nitrilase.

STATEMENT OF THE INVENTION

In brief, the method of this invention comprises partially cross-linkingenzymatically active microorganism cells or some other biologicalmaterial in aqueous solution or suspension through reaction withglutaraldehyde, followed by dewatering of the resulting flocculatedsolids, resulting in a pasty consistency mass of a (consistency) andcoherency suitable for particle shaping. Then, (wet) particles ofdesired shape and size are generated from the mixture followed bydrying. The polyazetidine prepolymer solution is incorporated before orafter dewatering, more preferably the former. Curing of thepolyazetidine prepolymer which occurs during the drying step, convertsthe pasty mass particles into cured particles of high physical strength.

Immobilized enzyme products prepared according to the invention employedin packed bed exhibit a very small pressure drop (e.g., a pressure dropwhich is only around 50% of a comparable prior art product) and a highphysical strength and resistance against abrasion, and moreover, theimmobilized enzyme products are relatively inexpensive to make due tothe high yield of usable particles. Also, it has been found thatpreferred embodiment immobilized enzyme product prepared according tothe invention exhibit a high volumetric activity.

DETAILED DESCRIPTION OF THE INVENTION

The cross-linking reactions between enzymatically active cellularsubstances and a polyazetidine prepolymer to crosslink the enzyme into acomposition capable of repeated use is taught by the above-referencedU.S. Pat. No. 4,436,813 and Paper of Wood et al., (see also G. J.Carlton et al, Biotechnology, vol. 4, pp. 317-320 (1986)) and,therefore, need not be discussed in detail here.

The polyazetidine polymer used for the practice of this invention may beany water-soluble polymer with a substantial content of reactiveazetidine rings, such as those prepared by reacting a polyamide withepichlorohydrin according to German patent publication No.DT-AS-1,177,8254. Examples of commercially available products arePolycup 2002, Kymene 557H and Reten 304 (these are all trade marks ofHercules Inc., U.S.A.).

A representative polyazetidine prepolymer in aqueous solution, such asis depicted below, e.g., Polycup 172 (Hercules, Inc.). ##STR1## iscross-linked by heat, H₂ O removal or pH adjustment to an alkaline pHvalue. Some of the curing reactions are reaction of the prepolymer withavailable --NH₂, --OH, --SH, --COOH groups of cellular substances on orfrom the enzymatically active microorganism cell.

Unless the reaction mass is sub-divided into individual particles priorto curing a cross-linked cell mass product will be a single block ofmaterial, i.e., a coherent mass that must then be sub-divided into thedesired particle from enzyme product. As has already been pointed out,such a conversion results in relatively low yield of the desired sizerange particles and, in addition, the shape of the individual particlesis not controlled at all.

Forming the cell mass into discrete particles prior to cross-linking ofthe polyazetidine prepolymer, would, of course, be desirable.Unfortunately, the fluid aqueous mixture of polyazetidine prepolymer andcell mass, e.g., cell sludge, is not well adapted to being formed intocoherent particles.

As has been pointed out, the improvement of this invention is directedto conversion of a biological material such as microorganism cells intoa wet past mass followed by subsequent subdivision of the mass intodiscrete particles. The present process can be considered to be apretreatment procedure that is carried out prior to curing thepolyazetidine. Subsequently, as the particles dry, the polyazetidinecross-linking reactions take place and the particles assume theirultimate coherency, hardness etc. Conversion, e.g., of the microorganismcells, into a relatively coherent pasty mass is accomplished by carryingout partial cross-linking of the biological material in aqueousdispersion by reaction with glutaraldehyde, followed by dewatering.

Biological materials that can be immobilized by the method of thisinvention include enzymes soluble or cell bound alike, microorganismcell (intact or disrupted cells, viable or non-viable), antibodies andcoenzymes. The biological material to be immobilized should be in anaqueous solution or dispersion and may have been purified as desired byconventional techniques. The degree of purification is not critical tothe practice of the invention.

It has been found that the quantity of water present in the(pretreatment) partial cross-linking reaction mixture of this inventionis not critical. Excess water will be removed from the flocculatedpartially cross-linked solid phase substance in the reaction productmixture during dewatering without any serious loss of active material.Thus, water may be added to the solution or dispersion of the biologicalmaterial to obtain a convenient consistency for the partialcross-linking reaction, then excess water is removed by dewatering. Theterm dewatering is employed herein within a context of physical removalof water, such as, for example, decanting, filtration, centrifugationand the like.

A convenient preferred starting material for practice of this inventionis the enzymatically active cell sludge recovered from a fermenterthrough filtering or centrifuging the culture broth. The cell sludge maybe used as such or first be homogenized. Since the fermenter may not bein close proximity to the immobilization facility, it is noted that theoptionally homogenized cell sludge may be stored in frozen state.Indeed, freezing, then thawing of the cell sludge maybe advantageousrather than be an activity losing detriment in the overall processsequence.

The detailed chemistry of the reactions involved in partialcross-linking through reaction of the microorganism cells and cellularsubstances with glutaraldehyde are not known to the inventors hereof.Indeed, insofar as the inventors hereof are aware, the chemistry ofcross-linking with glutaraldehyde is not fully elucidated. Reference ismade to Douglas J. Ford, 2. Reaction of Glutaraldehyde with Proteins,University of Cincinnati; and Hardy, The Nature of the Cross-linking ofProteins by Glutaraldehyde, Part I, Journal of the Chemical Society,Perkin Transactions, Vol. 1, Pg. 958, 1976. However, the art is familiarwith practical results from reacting glutaraldehyde with microorganismcells.

Glutaraldehyde has been suggested to the enzyme art for cross-linking togenerate cell mass particle form enzymes, as witness the teachings inthe aforementioned U.S. Pat. No. 3,980,521. Glutaraldehyde has also beensuggested for stabilizing cell bound enzymes on the microorganism cellsas witness the teachings in U.S. Pat. No. 3,779,869. The usage ofglutaraldehyde in practice of this invention is related to the teachingsof both above-referenced patents, yet is quite different therefrom.Although occurrence of cross-linking reactions is desired, in largemeasure the exact extent to which reaction with glutaraldehyde preventsloss of enzyme from individual microorganism cells (see U.S. Pat. No.3,779,869), or can convert cells and cell fragments into a coherentcovalently linked matrix (see U.S. Pat. No. 3,980,521), is not materialfor practice of this invention.

The purposes of treatment with glutaraldehyde in practice of thisinvention is generation of a reaction product mixture that containflocculated solids which then can be dewatered and so doing generates apasty mass adapted for subdivision into discrete particles. The pastymass is capable of admixture with an aqueous polyazetidine prepolymerand then be a mixture of a coherent consistency from which particles maybe formed. Generation of the particle forming capability is theobjective sought. It should be appreciated that the term "pasty mass" asemployed herein is both descriptive of the partially cross-linked (stillwet) dewatered product and connotes existence of cohesiveness and aparticle forming capability.

Incident to treatment of a cell sludge or other biologic material withglutaraldehyde, auxiliary cross-linking agents containing --NH₂ groupsmay be added to the reaction mixture e.g., polyethylene imine, chitosan,albumine, gelatine. Also, flocculating agents may be added. Further, itmay be advantageous to treat the cell sludge with a metal ion complexingagent such as EDTA. The exemplary details hereinafter provided aboutpreferred embodiments of this invention are not likely to be applicablein all their detail to other cell bound enzymes. Cut and try testswithin the preferred glutaraldehyde ranges are suggested to ascertainwhether inclusion of auxiliary cross-linking agents and/or inclusion ofpolymeric flocculating agents is advisable, or, perhaps, is necessary toachieve a workable consistency and the proper water content in thedewatering partially cross-linked cell mass.

It may be noted also that finely divided filler materials and/or enzymestabilizers (e.g., metal ions) when presence of such is desired in theultimate cell mass immobilized enzyme product may best be incorporatedinto the cell mass incident to the partial cross-linking withglutaraldehyde.

It has been found that the quantity of water in the cell sludge and thatadded with the glutaraldehyde and with any optional agent in the partialcross-linking reaction mixture such as flocculating agent, auxiliarycross-linking agent, enzyme stabilizer ions, etc. is not a criticalfactor. All water in excess will be filtered or centrifuged off from thepartially cross-linked pasty mass. However, the relative proportions ofcell sludge dry matter and glutaraldehyde are important.

According to one preferred mode of the invention, the polyazetidineprepolymer solution is incorporated into the pasty mass.

In such preferred embodiment of the method according to the invention,the amount of glutaraldehyde is between 5% and 40% w/w in regard to cellsludge dry matter, preferably between 10% and 20% w/w. If the amount ofglutaraldehyde is below 5% w/w, the filterability of the partiallycross-linked cell mass may be inferior, and if the amount ofglutaraldehyde is above 40% w/w, the enzyme yield recovery in theimmobilized enzyme product maybe unsatisfactory.

The dewatered partially cross-linked cell mass has a water content of70-90% w/w, preferably 80-85% w/w. If the water content is less than 70%w/w, the pressure drop characteristics of the particulate immobilizedenzyme product may not be satisfactory, and if the water content isabove 90% w/w, performance of the particle shaping step may beunsatisfactory. Thus, the relatively narrow 70%-90% water content in thedewatered cell mass is relatively critical in practice of the invention.

The practitioner of this invention will soon recognize the most workableconsistency area. The particle forming capability is somewhat poor atboth ends of the 70-90% water content range. It is noted that watercontent for the most workable consistency will vary enzyme to enzyme.

The water content range provided above for the dewatered partiallycross-linked cell mass takes into account that the polyazetidineprepolymer added will be as an aqueous solution of 10-15% solids. Thepreferred range of 80-85% water virtually assumes about a 12%polyazetidine prepolymer solution and the more preferred polyazetidineprepolymer content in regard to the dry weight of the cells will beemployed.

The polyazetidine prepolymer is added in an amount of between 5% and 30%w/w (dry matter basis), more preferably 10-20% w/w (dry matter basis) inregard to the cell sludge. If the amount is below 55% w/w, the pressuredrop reduction improvement obtained in the particulate product isunsatisfactorily low, and if the amount is above 30% w/w the enzymeyield in the immobilized enzyme particulate product is unsatisfactorilylow. Thus, should a particular cell-bound enzyme require less than 10%or more than 20% of the polyazetedine prepolymer for yield or productstability reasons, arbitrary adoption of the above given most preferred80-85% water content range is not advised.

It is to be understood that the foregoing discussion of proportions iswithin a context of incorporating the polyazetidine prepolymer solutioninto a partially cross-linked pasty mass. Such has not been found to benecessary, and a more preferred mode is to add the polyazetidine priorto dewatering, at all of which times the aqueous solution or dispersionof the biological material is still in fluid state.

Although, according to this mode of the invention, the polyazetidineprepolymer solution is added prior to dewatering, it has been found thatvery little of the polyazetidine is lost during dewatering. Apparentlypolyazetidine binds to the biological material, e.g., to enzymaticallyactive microorganism cells, by acting as a cationic flocculent, or thepolyazetidine can be made to bind to the material through selection ofappropriate flocculent(s). In any event, the need for a flocculent aswell as a suitable type and quantity of flocculent is readily determinedby those skilled in the art for whatever particular enzyme or otherbiological material is being cross-linked. Thus, optionally butdesirably, a flocculent is also added while the fluid state exists.

Aside from a requirement for a lesser amount of polyazetidine foroptimum product properties when polyazetedine prepolymer is added beforethe dewatering step, the process remains the same. The processingconditions for partial cross-linking with glutaraldehyde alreadydescribed apply, as for example temperature 0°-60° C., pH 5-9, buffer asneeded, cross-linking for 5-60 minutes, auxiliary crosslinking agentswhen advisable or when desired (to dilute the enzyme for instance). Theauxiliary cross-linking agents may be present in quantities of up to100% of the biological material by weight dry matter, preferably muchless, depending on the auxiliary agent, notably 20% or less forpolyethylene imine, 50% or less for albumin or gelatine and 10% or lessfor carboxymethyl cellulose. Inclusion of flocculant as appropriate.

The great advantage of incorporating the polyazetidine prepolymer intothe pre-dewatered and still fluid mixture is reduction in the proportionof polyazatidine required and for better control over the properties ofthe filter cake and of immobilized enzyme product.

Thus, a relatively high proportion of polyazetidine in the productgenerally yields physically strong particles, whereas a lower proportionyields particles with lower diffusion restriction and therefore withhigher activity. The most suitable amount of polyazetidine for this modeof the invention will usually be in the range about 0.1-about 10% byweight of total dry matter in the solution or dispersion, typicallyabout 0.5-5%, e.g., preferably about 0.5 to 3%.

In both modes of the invention discussed above, the partiallycross-linked dewatered mixture is a pasty mass that exhibits a coherencyand a consistency suited to particle shaping. A preferred particleshaping technique is to extrude the dewatered mixture, then partiallydry (drying cures the polyazetidine prepolymer), thereafter spheronize,followed by supplementary drying, the last being optional. Themarumerizing practice of Great Britain No. 1,362,265 may be followed.

Partially cross-linking the suspended biological material particleschanges their physical character, makes them sticky so that upondewatering of the two phase aqueous mixture the solids fuse into arelatively coherent pasty mass. Comparably. partially cross-linking adissolved biological material, such as for example enzyme in solution,causes precipitation of the biological material. Upon dewatering of thetwo phase aqueous mixture, the precipitate converts into a relativelycoherent pasty mass. Thus, in each event, dewatering, e.g., byfiltration, removes much of the water phase generating the desired pastymass. Prior to dewatering the precipitated solids or the partiallycross-linked particles which even collect in bunches, i.e., becomeflocculated. Inclusion of flocculant is to improve flocculation of thepartially cross-linked solid phase. Flocculants, when present andpolyazetedine prepolymer, when present, are mostly in the pasty mass.

To repeat the pasty mass made according to practice of this inventionexhibits a significant level of coherency, allowing the mass to besubdivided, e.g., extruded, and the extrudate ribbons do not fusetogether before curing of the polyazetidine prepolymer has beeneffected.

Additional water is removed during the curing step, e.g., by evaporationas the cross-linking reaction proceeds, so that a physically strongrelatively dry product is obtained. Preferred techniques are air dryingor fluidized-bed drying, generally at 15°-80° C. In case of verysensitive biological materials, low temperature drying or freeze-dryingmay be needed.

The pasty mass is a homogeneous mixture. Any particulate matter therein,e.g., finely, divided filler particles, is uniformly dispersed. Thecomposition of any one portion of the pasty mass will be no differentfrom any other portion. Such uniformity carries through to the productparticles. Each will have the same composition. Also, the center of eachparticle is of the same composition as the particle periphery. In thissense, the product particles are essentially homogeneous.

In all preferred embodiments of the method according to the invention,the particle form immobilized enzyme is dried to a water content ofabout 15-25% w/w. With an ultimate water content of above around 25% themicrobial stability of the product is unsatisfactory. Furthermore, aspreviously indicated, with water content above around 25% thecross-linking with the polyazetidine prepolymer may not have taken placeor may not be complete; the particles may tend to aggregate over time instorage. Drying to a water content of below about 15% may cause loss inenzyme activity.

UTILITY

The process of this invention is widely applicable to immobilization ofbiological materials, such as enzymes, particularly, glucose isomerase,penicillin acylase and nitrilase, cell mass, coenzymes and antibodies(monoclonal or polyclonal).

Enzymes which may be immobilized can be in the form of enzymaticallyactive cells, or partly or fully homogenized cell paste, or as a largelycell-free enzyme solution. Some instances where the method of theinvention is particularly advantageous are:

glucose isomerases from Streotomcyes sp.. e.g. from the followingspecies:

    ______________________________________                                        S. murinus                                                                    (EP 0 194 760)                                                                S. flavovirens                                                                S. achromogenus                                                               S. echinatus                                                                  S. wedmorensis                                                                S. albus                                                                      (U.S. Pat. No. 3,616,221)                                                     S. olivochromogenes                                                           S. venexueloe                                                                 (U.S. Pat. No. 3,622,463)                                                     S. griseoflavus                                                               (U.S. Pat No. 4,137,126)                                                      S. galbus                                                                     S. gracilis                                                                   S. margensis                                                                  S. niveus                                                                     S. plantensis                                                                 (Hungarian patent 12,415)                                                     S. violaceoniger                                                              (German patent 2,417,642)                                                     S. acidodurans                                                                (U.S. Pat. No. 4,399,222)                                                     S. phaeochromogenes                                                           S. fradiae                                                                    S. roseochromogenes                                                           S. olivoceus                                                                  S. californicus                                                               S. vanoceus                                                                   S. virginioe                                                                  (Japanese patent publication 69-28,473)                                       S. olivaceus                                                                  (U.S. Pat. No. 3,625,828)                                                     ______________________________________                                    

glucose isomerase from Bacillus coagulans. (see U.S. Pat. No.3,979,261).

glucose isomerase from Actinoplanes sp., especially A. missouriensis.

glucoamylase from Asperoillus sp., especially from black Aspergilli andmore especially from A. niger (see U.S. Pat. No. 3,677,902), or fromRhizoous sp.. especially Rh. delemar or Rh. niveus.

penicillin-V acylse from Fusarium sp., especially F. uioides, F.aroiilaceum, F. avenaceum, F. bulbioenum, F. coeruleum, F. eouiseti, F.lateritium, F. minimum, F. monoliforme, F. oxysporum, F. sambucinum, F.semisectum, F. solani, and F. sulphureum (see GB No. 891,173).

penicillin acylase from Eschericia coli, Proteus rettoeri, Kluyveracitrophila, Bacillus sohaericus. Bovista plumbea or Bacillus megaterium.

lactase from Kluyveromyces sp., especially from K. fragilis or K.lactis.

cyanide hydratase according to Danish DK No. 87/1283

nitrilase, nitrile hydratase or amidase, especially from Rhodococcussp., pseudomonas sp. or Brevibacterium sp.

See U.S. Pat. No. 4,001,081, EP Nos. 0 093 782 and 0 188 316.

The cell mass immobilized into cell mass particle form by the process ofthe invention may be viable. or non-viable intact cells as well as inthe form of homogenized cell paste. The cells are preferably ofmicrobial or plant orgin. Some preferred examples follow:

viable cells for use in bioconversion, e.g., yeast for ethanolfermentation.

cells with enzymatic activity, e.g., fungal mycelium containing cyanidehydratase, see EP Nos. 0 061 249 and 0 116 423.

cell mass preparations for use in adsorptive removal, e.g., of heavymetals, see EP No. 0 181 497, U.S. Pat. Nos. 4,320,093, 4,298,334,4,293,333 and JP-A No. 49-104,454.

EXEMPLARY APPLICATIONS OF THE INVENTION

As has already been pointed out, a preferred mode of this invention isdirected to instances when the art desires to convert cell bound enzymesinto a cell mass particle form. Within the above-described parametersfor practice of this invention is sufficient variability to makepractice of the invention applicable to any microorganism source cellbound enzyme. For example, glucose isomerase a well known cell boundenzyme, has been produced on a large scale cultivation of Bacilluscoagulans. An excellent glucose isomerase is elaborated by glucoseisomerase producing strain belonging to the genus Streotomcyes, e.g., aStreotomcyes murinus cluster strain. The cell bound enzyme from Bacilluscoaoulans can be immobilized readily by reaction with glutaraldehyde;see the aforementioned U.S. Pat. No. 3,980,521. However, the cell boundenzyme produced by strains of the genus Streptomyces, including notablythe Streptomyces murinus cluster is characterized by an insufficientcross-linking capability with glutaraldehyde to produce a satisfactorycell mass particle form immobilized enzyme. However, either of thesecell bound enzymes may be immobilized in cell mass particle form throughpractice of this invention. It follows, of course, that practice of thisinvention is particularly suited to the Streptomyces murinus enzyme.Glucose isomerase is a commercially important enzyme, which is to say,that immobilization of the Streptomyces murinus glucose isomerase is onepreferred mode practice of this invention.

The widespread applicability of this invention is also exemplifiedhereinafter by a disparate commercially important cell bound enzyme,i.e., trytophan synthetase derived from a strain of E. coli. Preparationof this enzyme, too, is a preferred mode practice of the invention. Noneof the prior art immobilization methods investigated by the inventorshereof resulted in an immobilized tryptophan synthetase product ofsatisfactory physical strength. It is noted that tryptophan synthetaserequires a cofactor. Such is present in the cytoplasm. Since disruptionof the cell would cause loss of the cofactor, immobilization of wholecells is employed in the instance of the tryptophan synthetase enzyme.

Enzyme granules made according to practice of this invention,particularly according to preferred practices of the invention, exhibitsuperior physical properties. In packed bed they exhibited a pressuredrop for the liquid flowing therethrough which is only around 50% of acomparable prior art product. (In this test study, the comparable priorart product was a glutaraldehyde cross-linked Bacillus coaoulans glucoseisomerase made according to the teachings of U.S. Pat. No. 3,980,521, aproduct that is in widespread commercial usage.) In addition, highphysical strength and resistance against abrasion were found.

For further understanding of the practice of this mode of the invention,the following specific Examples are presented.

EXAMPLE 1

Glucose isomerase containing cells of Streptomyces murinus, DSM 3252were cultivated in a conventional medium comprising glucose, a complexnitrogen source, minerals and trace elements.

After pH adjustment of 7.0-7.5 the cells were recovered from thefermentation broth by centrifugation and homogenized after addition ofMgSO₄, 7H₂ O in an amount of 0.5% w/v by means of a Manton-Goulinhomogenizer. The homogenized cell sludge was kept in -18° C. and thawedimmediately before use in a immobilization experiment.

300 g of homogenized cell sludge with a dry matter content of 6.7% wasdiluted to 750 ml with 1.5% MgSO₄, 7H₂ O, and pH was adjusted to 7.5. 30g Corcat p-18 polyethylene imine flocculent (Cordova Chem. Co.) wasadded. Then 9.24 g of 50% glutaraldehyde was added; pH was maintained at7.4-7.6 for one hour. The flocs were collected by filtration.

The filter cake containing approximately 15.8% DM was divided into twoequal parts in terms of dry matter content. The two parts were mixedwith 0 and 15% w/w (dry matter basis) respectively of Polycup® 1884polyazetidine prepolymer solution, pH 7.5 (from Hercules, Inc.,Delaware) calculated on filter cake dry matter. Both parts were extrudedthrough 0.8 mm orifices. The extruded material was allowed to dry atroom temperature to a dry matter content of 83-85% w/w. The 300-700 ufraction was obtained by sieve fractionation.

The glucose isomerase activity (measured according to NOVO Document F850399) recovered in the two immobilized enzyme preparations wasapproximately the same. Pressure drop (in g/cm2) measured according toNOVO, AF 166 is given in the following Table 1.

                  TABLE 1                                                         ______________________________________                                        Pressure drop versus percentage of polyazetidine,                             calculated as dry matter on filter cake dry matter.                           ______________________________________                                        % polyazetidine     0       15                                                Pressure Drop (25 h/50 h)                                                                         14/17   6/7                                               ______________________________________                                    

EXAMPLE 2

A tryptophan synthetase producing strain of E. coli, ATCC 15491, wasgrown on an agar slant at 37° C. and from there transferred to apreculture in shake flasks at 37° C. The preculture was inoculated on amedium prepared as follows. The composition of the medium was:

    ______________________________________                                        (NH.sub.4).sub.2 SO.sub.4                                                                            8      g/l                                             KH.sub.7 PO.sub.4      1.6    g/l                                             Na.sub.2 HPO.sub.4.2H.sub.2 O                                                                        5.6    g/l                                             Trisodiumcitrate, 2H.sub.2 O                                                                         0.5    g/l                                             NaCl                   3      g/l                                             MgSO.sub.4.7H.sub.2 O  0.5    g/l                                             CaCl.sub.2.2H.sub.2 O  00.2   g/l                                             FeCl.sub.3 2H.sub.2 O  90     mg/l                                            ZnSO.sub.4.7H.sub.2 O  20     mg/l                                            MgSO.sub.4.4H.sub.2 O  24     mg/l                                            MnSO.sub.4.4H.sub.2 O  22     mg/l                                            CuSO.sub.4.5H.sub.2 O  4      mg/l                                            KI                     4      mg/l                                            NaMoO.sub.4.2H.sub.2 O 4      mg/l                                            H.sub.3 BO.sub.3.6H.sub.2 O                                                                          1.2    mg/l                                            CoCl.sub.2.6H.sub.2 O  6      mg/l                                            NiCl.sub.2.6H.sub.2 O  6      mg/l                                            *Biotin                4      μg/l                                         *Calcium pantothenate  800    μg/l                                         *Folic acid            4      μg/l                                         *Inositol              4000   μg/l                                         *Niacin                800    μg/l                                         *p-aminobenzoic acid   400    μg/l                                         *Pyridoxine HCl        800    μg/l                                         *Riboflavin            400    μg/l                                         *Thiamine HCl          800    μg/l                                         ______________________________________                                    

The medium was sterilized at 121° C. for 25 minutes except for thevitamins (*), which were added by sterile filtration after coolingtogether with dextrose (10 g/l) and indole in 48% ethanol (125 mg/l).The submerged fermentation was conducted under aseptic conditions at 37°C. with aeration at 1 volume/volume/minute, agitation at 500 rpm and pHcontrolled by acid/base addition at pH 7.0 for 40 hours. 24 hours afterinoculation a further addition of dextrose (40 g/l) and indole in 48%ethanol (875 mg/l) was carried out. Cells were harvested after 40 hoursby centrifugation and immediately used for the immobilizationexperiments described as Example 3 hereinafter.

EXAMPLE 3

60 g of wet cells recovered as described in Example 2, having a drymatter content of 17% w/w were resuspended in 1200 ml of 0.2M EDTA(adjusted to pH 7.5) and left for 30 min at room temperature and thencentrifuged. The aqueous EDTA washing procedure was repeated once.

The wet cells were mixed with 300 of 85 mM KH₂ P₄, pH 7.5; 8.4 g ofpolystyrene from Kodak (200-400 mesh, cross-linked with 2% w/wdivinylbenzoic acid), 96 mg of pyridoxal phosphate and 9.6 g of a 12.5w/v glutaraldehyde solution. The pH value was maintained at 7.5 byaddition of base throughout the immobilization procedure. Then 60 g ofCorcat P-150 polyethylene imine solution from Cordova Chemical Companyof Michigan, adjusted to pH 7.5 with 10N NaOH, was added, followed bythe same amount of glutaraldehyde solution as before. Dilution wascarried out with 900 ml of 50 mM KH₂ PO₄, pH 7.5 after 1 hour from thefirst addition of glutaraldehyde. Then flocculation was performed byaddition of 250 ml of 1% w/v Superloc A 130 from American Cyanamide.Partially cross-linked and flocculated cells were recovered byfiltration. The filter cake containing approximately 19% w/w of drymatter was divided into two equal parts. One part was mixed with 7.2 gof Polycup® 172 (Hercules, Inc., Delaware) pH 7.5, corresponding to16.5% w/w of dry polyazetidine prepolymer in regard to the dry matter inthe wet cells recovered by centrifugation from the fermentation.

Both parts were extruded through 0.8 mm holes.

The extruded material (both parts) was allowed to dry at roomtemperature to a dry matter content of 90% w/w. The 300-700 um fractionwas obtained by sieve fractionation.

The tryptophan synthetase activity recovered in the two immobilizedenzyme product was approximately the same. The pressure drop (in g/cm2)measured according to NOVO AF 166 of the product with polyazetidine wa14(25 h)/15 (50 h) and of the product without polyazetidine 23 (25 h)/24(50 h).

The examples which now follow are directed to the more preferred mode ofthe invention wherein the polyazetidine prepolymer is added prior todewatering, including being present during the partial cross-linkingtreatment. All other (dry substance) ingredients also are present in thepretreatment reaction mixture.

It may now be appreciated better that the underlying rationale to thismore preferred mode of practice of this invention is to achieveconversion of an enzyme (or other biologic material) in aqueous solutionor uniform dispersion together with any other dissolved or dispersedingredients into a two phase mixture wherein desirably the (partiallycross-linked) solid phase will contain all of the (dry basis) substancesdesired in the final product and the aqueous phase nothing but water andundesired ions, etc. Dewatering converts the solid phase substances intoa pasty mass for forming into coherent particles. Thus, flocculants,auxiliary cross-linking agent and other optional ingredients are addedto the aqueous solution or dispersion when their presence facilitatesgeneration of a more suitable two phase dewaterable inhomogeneousmixture.

The examples which follow exemplify most preferred practice of theinvention and in addition illustrate the effect of varying theingredients in the (partial crossinking) reaction mixture so as to besuggestions to those skilled in the art how best to approachimmobilizing some biological material not exemplified herein.

EXAMPLE 4

Glucose isomerase containing cells were produced by fermentation ofStreptomyces murinus, strain DSM 3253, according to Example 1.

The cells were harvested by centrifugation of the culture broth. Thecell sludge had a dry substance content of 7.0%.

The general immobilization procedure was as follows; To 300 g cellsludge was added 300 g deionized water containing 1.5% MgSO₄, 7H₂ O. pHwas adjusted to 7.5. The indicated amount of polyethyleneimine (Sedipur,product of BASF, West Germany) was added, and after thorough mixing themixture was cross-linked by addition of 15% active glutaraldehyde basedon cell sludge dry substance plus polyethyleneimine dry substance. After1 hour the polyazetidine prepolymer (Polycup ®2002) was added andthoroughly mixed with the cross-linked cell suspension.

The mixture was then flocculated by addition of a cationic flocculent,Superfloc C521 (Cyanamid Int.). The cross-linked enzyme was recovered byfiltration, formed into particles by extrusion through a 0.8 mm screenand dried at room temperature.

The glucose isomerase activity was measured by NOVO analysis methodF-855310 (available on request from Novo Industri A/S, Denmark) and thephysical stability determined as pressure drop over a column.

The pressure drop was measured over a column with a diameter of 24 mmand an enzyme bed height of 4 cm (5 g enzyme). The solution, 45% glucosein demineralized water with 1 g MgSO₄ S/1, was pumped through the columnat a rate of 40 g/min at 60° C. The pressure drop (in mm of liquid)describes the physical stability of the enzyme particle, i.e., a lowpressure drop corresponds to a good physical stability. The results areshown in the table below.

    ______________________________________                                                     Glucose isomerase                                                                          Pressure drop                                                    activity (μmol/min/g)                                                                   (mm)                                                ______________________________________                                        5%      0% poly-   614            400                                         poly-   azetidine                                                             ethylene-                                                                             2.5% poly- 667            99                                          imine   azetidine                                                                     5% poly    586            57                                                  azetidine                                                             10%     0% poly-   692            105                                         poly-   azetidine                                                             ethylene-                                                                             2.5% poly- 578            11                                          imine   azetidine                                                                     5% poly    506            10                                                  azetidine                                                             ______________________________________                                    

Preparations with pressure drops exceeding about 20 mm liquid are notconsidered to be well suited to industrial glucose isomerase columns.Activity decreases slightly with increasing polyazetidineconcentration.,

This example clearly shows the improvement of physical stability of theimmobilized preparations obtained with polyazetidine.

EXAMPLE 5

A similar experiment to that described in Example 4 was performed usinga higher yielding descendant of DSM 3253.

The dry substance content of the cell sludge was 5.3%, and the cellswere partially disrupted by homogenization. Otherwise the immobilizationwas performed as described in Example 4.

The results are given in the Table below.

    ______________________________________                                                     Glucose isomerase                                                                          Pressure drop                                                    activity (μmol/min/g)                                                                   (mm)                                                ______________________________________                                        5%      0% poly-   556            16                                          poly-   azetidine                                                             ethylene-                                                                             1% poly-   855            13                                          imine   azetidine                                                                     2.5% poly- 839            13                                                  azetidine                                                                     5% poly-   707             8                                                  azetidine                                                             10%     0% poly-   651            12                                          poly-   azetidine                                                             ethylene-                                                                             1% poly-   975            10                                          imine   azetidine                                                                     2.5% poly- 911             5                                                  azetidine                                                                     5% poly-   853             3                                                  azetidine                                                             ______________________________________                                    

From these experiments, it can be concluded that the polyazetidineimproves the physical stability of even very physically stableformation.

EXAMPLE 6

Bacillus coagulans containing glucose isomerase was immobilized with andwithout polyazetidine.

Homogenized cell paste of B. coaoulans prepared according to U.S. Pat.No. 3,979,261 was suspended in 0.1% MgSO₄ to a final dry substanceconcentration of 3%. Glutaraldehyde was added to a final concentrationof 0.5%. After 60 min with mixing at room temperature polyazetidineprepolymer (Polycup® 2002) wa added followed by floccillation withSuperfloc C521. The cross-linked enzyme was recovered by filtration,formed into particles by extrusion and dried at room temperature.

Activity and pressure drop were measured as described in Example 1.Resistance to grinding was measured as turbidity (optical density) at600 nm after 1 hour of vigorous stirring of 0.5 g enzyme in 20 ml 50 mMphosphate, pH 7 with a propeller. Before the assay the enzyme producthad been swelled and washed in 6% NaCl in 50 mM phosphate, pH 7.0.

    ______________________________________                                        Amount of         0        1        3                                         polyazetidine (% dry substance)                                               activity (μmole/min/g)                                                                       540      506      493                                       pressure drop (mm liquid)                                                                       5        3        2                                         25 grinding       0.111    0.068    0.062                                     ______________________________________                                    

Both resistance to grinding and physical stability were improved byadding polyazetidine, while only a small activity loss was observed.

EXAMPLE 7

To exemplify immobilization of a soluble enzyme the amyloglucosidasefrom Aspergillus niger has been chosen. This enzyme is extracellular.

A commercial preparation AMG 400 L HP (Novo Industri A/S, Denmark) wasdialyzed against 50 mM phosphate pH 7.0. The preparation was diluted toa dry substance concentration of 2% w/v, and an equal ammount of eggalbumen was added (i.e., 2% w/v). Glutaraldehyde was added to aconcentration of 0.6% w/v. After one hour of stirring, polyazetidineprepolymer (Polycup^(R) 2002) was added to a final concentration of0.08% w/v, and the mixture was flocculated with Filtrafloc (Servo B.V.,Netherlands). Enzyme was recovered as described in previous examples.

Activity was measured as described in Novo Analysis Method AF159/2.Pressure drop was measured as described in Example 1, but at 35° andwith 11% w/w glucose in 50 mM phosphate, pH 7.5.

    ______________________________________                                        polyazetidine, % w/v 0         0.08                                           activity (μmol/min/g)*                                                                          196       160                                            20 pressure drop (mm liquid)                                                                       9         6                                              ______________________________________                                         *Particle fraction: 425-710 μm                                        

EXAMPLE 8

Cell paste of Fusarium sp. containing penicillin acylase activity(prepared according to British patent specification No. GB 891,173),which had been washed thoroughly with 0.9% NaCl, was suspended in 50 mMphosphate pH 7.0 to give a final dry substance content of 3%.Polyethyleneimine (Sedipur) was added to give a final dry substancecontent of 0.1% Glutaraldehyde was added to a final concentration of0.2% w/v. After one hour with thorough mixing polyazetidine prepolymerwas added, and finally the mixture was flocculated with a cationicflocculent Filtrafloc. The cross-linked enzyme was recovered byfiltration, formed into particles by extrusion through a 0.6 mm screenand dried at room temperature.

Enzyme activity was measured by Novo analysis AF186, and the physicalstability determined as pressure drop and resistance to grinding (seeExample 6).

    ______________________________________                                                                           Kymene ®                               Polyazetidine type                                                                         None    Polycup ® 2002                                                                          557H                                       ______________________________________                                        Polyazetidine (%)                                                                          0       1        3      1                                        activity* (PVU/g)                                                                          72      57       42     35                                       10 pressure drop (mm                                                                       6       4        3      2                                        liquid)                                                                       grinding     0.563   0.305    0.140  0.103                                    ______________________________________                                         *450-710 μm fraction                                                  

As can be seen from the above Table polyazetidine increases physicalstability, i.e., gives increased resistance to grinding and gives highlyimproved pressure stability. However, this improvement is obtained atthe expense of an activity loss which partly is due to diffusionlimitation, which again is believed to be due to a more densepreparation when polyazetidine is present.

EXAMPLE 9

Fusarium sp. was immobilized as described in Example 5, but at differentpH-values and with 1% Kymene® 557H in all preparations.

Results are seen in the table below:

    ______________________________________                                        pH               6         7       8                                          25 activity (PVU/g)                                                                            33.3      36.1    41.5                                       pressure drop (mm liquid)                                                                      2         3       3                                          grinding         0.171     0.154   0.188                                      ______________________________________                                    

The physical stability is good and independent of pH in the tested range(6-8).

EXAMPLE 10

The significance of time of polyazetidine prepolymer addition wasexamined with cell paste of Fusarium sp. Immobilization was done asexplained in Example 5, but without PEI and with polyazetidineprepolymer (Polycup® 2002) addition to a final concentration of 0.3%both before and after the addition of glutaraldehyde.

    ______________________________________                                        Time for polyazetidine                                                                     not     before      after                                        addition     added   glutaraldehyde                                                                            glutaraldehyde                               activity (PVU/g)                                                                           58      32          31                                           10 pressure drop                                                                           27       6           4                                           (mm liquid)                                                                   ______________________________________                                    

The physical stability is increased both when polyazetidine is addedbefore and after the glutaraldehyde.

EXAMPLE 11

Cell sludge of Rhodococcus erythropolis having ability to hydroyzenitrile (prepared according to EP No. 188,316) was diluted to 2% drysubstance with water. 10% polyethyleneimine (Sedipur) (based on drysubstance of cell sludge) was added, and pH was adjusted to 7.0. Themixture was cross-linked by addition of 5% active glutaraldehyde (basedon cell sludge dry substance plus polyethyleneimine dry substance).After 1 hour, 2% of polyacetidine prepolymer (Kymene) (based on totaldry substance) was added.

The mixture was then flocculated by addition of an anionic flocculent,Superloc A 130. The cross-linked enzyme was then recovered byfiltration, formed into particles by extrusion through a 0.8 mm screenand dried at room temperature.

For reference, the same cell sludge was cross-linked withoutpolyazeteidine, but otherwise in the same way as above.

For comparison, the same cell sludge was immobilized by a prior-artmethod, viz. entrapment in polyacrylamide gel according to U.S. Pat. No.4,248,968. More specifically, the cell sludge was diluted to 12.5% drysubstance and immobilized as described in Example 12 thereof. Finally,the gel was sieved with 1 mm mesh sieve and washed with saline until thesupernatant became clear.

    ______________________________________                                        11% glucose syrup                                                             ______________________________________                                        Flow rate:     40 g/min                                                       Temperature:   35° C.                                                  ______________________________________                                    

Pressure drop was measured after 25 hours.

5 g (dry substance) of immobilized enzyme was applied for pressure dropmeasurement.

    ______________________________________                                        Results (pressure drop in g/cm.sup.2):                                        ______________________________________                                        Invention                                                                     cross-linking with polyazetidine                                                                    13                                                      Reference                                                                     cross-linking without polyazetidine                                                                 >200                                                    gel entrapment        >200                                                    ______________________________________                                    

The practical effect of physical stability of a product is illustratedby the fact that a product with a pressure drop (mm liquid) of 2-4 canbe used in a fixed bed column reactor with a height of 1 meter and adiameter of 1 meter with a holding time for the liquid passingtherethrough of 1 minute. The packed bed may be functioning with aconstant pressure drop over the bed for more than 6 months. A productwith a pressure drop of more than 10 is not able to withstand theseconditions.

The same situation is also seen when the process takes place in acontinuous stirred tank system. Products with low grinding propertiesare significantly more stable in e.g. 4 m² reactor systems. Practicallifetimes of optimal products of more than 4 months can be expectedwithout more loss than 50% activity.

We claim:
 1. A process for forming polyazetidine cross-linkedimmobilized biologically active materials in particle form whichconsists essentially of:partially cross-linking an aqueous dispersion orsolution of a biologically active material with glutaraldehyde, toproduce a two phase system of flocculated partially cross-linked solidscontaining said biologically active material and water, dewatering saidtwo phase system and recovering the solids phase as a wet pasty mass,sub-dividing said pasty mass into discrete particles each of which isessentially homogeneous adding a polyazetidine prepolymer before, or atthe beginning of partially cross-linking or subsequent thereto but priorto subdividing said pasty mass into particles, and thereafter curingsaid particles whereby said polyazetidine prepolymer undergoescross-linking.
 2. The process of claim 1 wherein the glutaraldehydecontent in said dispersion or solution comprises 5-40% by weight of thebiological material.
 3. The process of claim 1 wherein saidpolyazetidine prepolymer is added to said aqueous dispersion prior toinstituting said partial cross-linking.
 4. The process of claim 3wherein the polyazetidine is 0.5-5% w/w of total dry matter.
 5. Theprocess of claim 1 wherein said polyazetidine prepolymer is added aftersaid partial cross-linking but prior to dewatering.
 6. The process ofclaim 5 wherein the polyazetidine is 0.5-5% w/w of total dry matter. 7.The process of claim 1 wherein said pasty mass is mixed with aqueousprepolymer.
 8. The process of claim 7 wherein the polyazetidine is10-20% w/w dry matter basis of the biologically active material.
 9. Theprocess of claim 1 wherein water content of said particles is reduced tobelow about 25% by weight during the curing thereof.
 10. The process ofclaim 1 wherein said biologically active material is an enzyme.
 11. Theprocess of claim 10 wherein said enzyme comprises enzymatically activewhole, fragmented or homogenized microorganism cells.
 12. The process ofclaim 11 wherein the microorganism is a strain from a species selectedfrom the genus group consisting of Streptomyces, Bacillus, Actinoplanes,Fusarium, Rhodococcus, Pseudomonas and Brevibacterium.
 13. The processof claim 11 wherein said enzyme comprises cell bound glucose isomerase.14. The process of claim 10 wherein said enzyme is selected from thegroup consisting of glucose isomerase, penicillin acylase and nitrilase.15. The process of claim 10 wherein said enzyme is in solution whenpartial cross-linking begins.
 16. The process of claim 1 wherein aflocculating agent is added to said dispersion or solution before,during or after beginning said partial cross-linking, but prior to saiddewatering.
 17. The process of claim 1 wherein partially cross-linkingsaid biologically active material is carried out in the presence of oneor more auxiliary cross-linking agents selected from the groupconsisting of polyethylene imine, gelatine, albumin and carboxymethylcellulose.
 18. An immobilized enzyme product made according to theprocess of claim 10.